Aerobic hydrogenase activity in Anacystis Nidulans The oxyhydrogen reaction

1. The oxyhydrogen reaction of Anacystis nidulans was studied manometrically and polarographically in whole cells and in cell-free preparations; the activity was found to be associated with the particulate fraction.2. Besides O₂, the isolated membranes reduced artificial electron acceptors of positive redox potential; the reactions were unaffected by O₂ levels <10–15%; aerobically the artificial acceptors were reduced simultaneously with O₂.3. H₂-supported O₂ uptake was inhibited by CO, KCN and 2-n-heptyl-8-hydroxyquinoline-N-oxide. Inhibition by CO was partly reversed by strong light. Uncouplers stimulated the oxyhydrogen reaction.4. The kinetic properties of O₂ uptake by isolated membranes were the same in presence of H₂ and of other respiratory substrates.5. Low rates of H₂ evolution by the membrane preparations were found in presence of dithionite; methyl viologen stimulated the reaction.6. The results indicate that under certain growth conditions Anacystis synthesizes a membrane-bound hydrogenase which appears to be involved in phosphorylative electron flow from H₂ to O₂ through the respiratory chain.     

Intracellular Location and O2 Sensitivity of Uptake Hydrogenase in Azospirillum spp

Uptake hydrogenase activity of Azospirillum brasilense in vitro (cell-free extract) was very much more sensitive to O₂ than was that of A. amazonense, and the O₂ pressure optima for uptake hydrogenase activities were 0.01 and 0.4 to 3 kPa for A. brasilense and A. amazonense, respectively. The addition of superoxide dismutase did not increase uptake hydrogenase activity of A. brasilense either in vivo or in vitro. The O₂ uptake rates of A. brasilense and A. amazonense were nearly the same. Inhibition of A. brasilense O₂-dependent uptake hydrogenase activity by O₂ was highly reversible under the conditions tested. O₂ also markedly inhibited in vitro methylene blue-dependent uptake hydrogenase activity of A. brasilense, and this inhibition was highly reversible. It is concluded that the difference in O₂ tolerance of the uptake hydrogenases is not due to a difference in respiratory protection in the two species and may be due to inherent differences in the two enzymes. For the three species, A. brasilense, A. amazonense, and A. lipoferum, almost all the recovered methylene blue-dependent uptake hydrogenase activity was associated with the membrane fraction.     

Periplasmic Cytochrome c3 of Desulfovibrio vulgaris Is Directly Involved in H2-Mediated Metal but Not Sulfate Reduction

Kinetic parameters and the role of cytochrome c₃ in sulfate, Fe(III), and U(VI) reduction were investigated in Desulfovibrio vulgaris Hildenborough. While sulfate reduction followed Michaelis-Menten kinetics (K_m = 220 μM), loss of Fe(III) and U(VI) was first-order at all concentrations tested. Initial reduction rates of all electron acceptors were similar for cells grown with H₂ and sulfate, while cultures grown using lactate and sulfate had similar rates of metal loss but lower sulfate reduction activities. The similarities in metal, but not sulfate, reduction with H₂ and lactate suggest divergent pathways. Respiration assays and reduced minus oxidized spectra were carried out to determine c-type cytochrome involvement in electron acceptor reduction. c-type cytochrome oxidation was immediate with Fe(III) and U(VI) in the presence of H₂, lactate, or pyruvate. Sulfidogenesis occurred with all three electron donors and effectively oxidized the c-type cytochrome in lactate- or pyruvate-reduced, but not H₂-reduced cells. Correspondingly, electron acceptor competition assays with lactate or pyruvate as electron donors showed that Fe(III) inhibited U(VI) reduction, and U(VI) inhibited sulfate loss. However, sulfate reduction was slowed but not halted when H₂ was the electron donor in the presence of Fe(III) or U(VI). U(VI) loss was still impeded by Fe(III) when H₂ was used. Hence, we propose a modified pathway for the reduction of sulfate, Fe(III), and U(VI) which helps explain why these bacteria cannot grow using these metals. We further propose that cytochrome c₃ is an electron carrier involved in lactate and pyruvate oxidation and is the reductase for alternate electron acceptors with higher redox potentials than sulfate.     

Autotrophic growth of strains ofRhizobium and properties of isolated hydrogenase

Four strains ofRhizobium (R. trifolii RCL10,R. japonicum S19 and SB16, andRhizobium sp. NEA4) were demonstrated to grow lithoautotrophically with molecular hydrogen as sole electron donor and with ammonium or with N₂ as N source. All of them showed ribulose-1,5-bisphosphate carboxylase activity and hydrogenase (H₂-uptake) activity with methylene blue and oxygen as electron acceptors. ForR. japonicum SB 16, a doubling time under autotrophic conditions of 30 h and a specific hydrogenase activity (methylene blue reduction) in crude extracts of 1.4 U/mg protein were calculated.Rhizobium hydrogenase is a membrane-bound enzyme. It is mainly detectable in particulate cell fractions, it cross-reacts with the antibodies of the membrane-bound hydrogenase ofAlcaligenes eutrophus, and is unable to reduce NAD. The isolated hydrogenase is a relatively oxygen-sensitive enzyme with a half-life of three days when stored at 4°C under air.     

Autotrophic growth of nitrogen-fixingAzospirillum species and partial characterization of hydrogenase from strain CC

Out of 15 strains ofAzospirillum spp. isolated from the roots of different plants, only 4 (CY, M, CC, and AM) were able to grow autotrophically with H₂ and CO₂. All of them showed H₂ uptake in the presence of oxygen or methylene blue and ribulose-1,5-bisphosphate carboxylase activity. Among the four strains, strain CC isolated from the roots ofCenchrus cilliaris showed maximum H₂+O₂ uptake (32.5 l/min. mg protein) as well as H₂ uptake in the presence of methylene blue (41.4 l/min·mg protein) and also the maximum activity of ribulose-1,5-bisphosphate carboxylase (17 units [U]/g protein). The doubling time of this strain under autotrophic growth conditions and at low oxygen concentration (2.5%, vol/vol) was 10 h. At the same O₂ concentration the maximal rates of H₂+O₂ uptake were reached. The distribution of hydrogenase activity among soluble and particulate protein fractions revealed that the hydrogenase ofAzospirillum strain CC is a membrane-bound enzyme. It showed cross-reaction with antibodies raised against the membrane-bound hydrogenase ofAlcaligenes eutrophus. The hydrogenase in intact cells and crude extracts reacted with methylene blue, phenazine methosulfate, and ferricyanide, but not with NAD or FMN. The specific hydrogenase activity, with methylene blue as an acceptor, was 5.71 U/mg protein in crude extract at 9.38 U/mg protein in the membrane suspension. Hydrogen evolution from reduced viologen dyes could not be demonstrated. The hydrogenase is oxygen sensitive and can be optimally stabilized by addition of dithionite to H₂-gased samples.     

Light-inducible Cytochrome Reduction in Membrane Preparations from Corn Coleoptiles: I. STABILIZATION AND SPECTRAL CHARACTERIZATION OF THE REACTION

Conditions for obtaining reproducible light-induced reduction of a b-type cytochrome in membrane fractions from coleoptiles of dark-grown Zea mays L. include a glucose-glucose oxidase system that lowers O₂ tension and generates H₂O₂, substrate amounts of ethylenediaminetetraacetic acid which, in some manner, facilitates photoreduction by both added flavin and the endogenous photoreceptor and a sample temperature below 10 C. Cytochrome reduction could be obtained by photoexcitation of either a tightly bound endogenous receptor, which is probably a flavin, or added riboflavin, flavin mononucleotide, or flavin adenine dinucleotide. The latter flavin was the least effective. The endogenous photoreceptor appears to be rather firmly bound to the membranes, suggesting that this association may also exist in vivo. When any of the above four photoreceptors or methylene blue were used to sensitize the reaction, a cytochrome with a reduced α-band near 560 nanometers and a Soret difference peak near 429 nanometers was the electron acceptor. This cytochrome could be clearly distinguished spectrally from other cytochromes that predominated in the membrane preparations.     

Characteristics of the H2 oxidizing system in soybean nodule bacteroids

A derivative of Rhizobium japonicum (strain 122 DES) has been isolated which forms nodules on soybeans that evolve little or no H₂ in air and efficiently fixes N₂. Bacteroids isolated from nodules formed by strain 122 DES took up H₂ with O₂ as the physiological acceptor and appeared to be typical of those R. japonicum strains that possess the H₂ uptake system. The hydrogenase system in soybean nodules is located within the bacteroids and activity in macerated bacteroids is concentrated in a particulate fraction. The pH optimum for the reaction is near 8.0 and apparent K _m values for H₂ and O₂ are 2 M and 1 M, respectively. The H₂ oxidizing activity of a suspension of 122 DES bacteroids was stable at 4°C for at least 4 weeks and was not particularly sensitive to O₂. Neither C₂H₂ nor CO inhibited O₂ dependent H₂ uptake activity.Non-physiological electron acceptors of positive oxidation reduction potential also supported H₂ uptake by bacteroids. The rate of H₂ uptake with phenazine methosulfate as the acceptor was greater than that with O₂. When methylene blue, triphenyltetrazolium, potassium ferricyanide or dichlorophenolindophenol were added to bacteriod suspensions, without preincubation, rates of H₂ uptake were supported that were lower than those in the presence of O₂. Preincubation of the bacteroids with acceptors increased the rates of H₂ uptake. No H₂ evolution was observed from reaction mixtures containing bacteroid suspensions and reduced methyl or benzyl viologens. Of a series of carbon substrates added to bacteroid suspensions only acetate, formate or succinate at concentrations of 50 mM resulted in 20% or greater inhibition of H₂ oxidation.The H₂ uptake capacity of isolated 122 DES bacteroids (expressed on a dry bacteroid basis) was at least 10-fold higher than the rate of the nitrogenase reaction in nodules expressed on a comparable basis. Since about 1 mol of H₂ is evolved for every mol of N₂ reduced during the N₂ fixation reaction, these observations explain why soybean nodules formed by strain 122 DES and other strains with high H₂ uptake activities have a capacity for recycling all the H₂ produced from the nitrogenase reaction.Abbreviations PMS PHenazine methosulfate - MB Methylene blue     

Anaerobic hydrogenase activity in Anacystis nidulans H2-dependent photoreduction and related reactions

1. Anaerobic hydrogenase activity in whole cells and cell-free preparations of H₂-induced Anacystis was studied both manometrically and spectrophotometrically in presence of physiological and artificial electron acceptors.2. Up to 90% of the activity measured in crude extracts were recovered in the chlorophyll-containing membrane fraction after centrifugation (144 000 × g, 3 h).3. Reduction of methyl viologen, diquat, ferredoxin, nitrite and NADP by the membranes was light dependent while oxidants of more positive redox potential were reduced also in the dark.4. Evolution of H₂ by the membranes was obtained with dithionite and with reduced methyl viologen; the reaction was stimulated by detergents.5. Both uptake and evolution of H₂ were sensitive to O₂, CO, and thiol-blocking agents. The H₂-dependent reductions were inhibited also by the plastoquinone antagonist dibromothymoquinone, while the ferredoxin inhibitor disalicylidenepropanediamine affected the photoreduction of nitrite and NADP only. 3-(3,4-Dichlorophenyl)-1,1-dimethylurea did not inhibit any one of the H₂-dependent reactions.6. The results present evidence for a membrane-bound ‘photoreduction’ hydrogenase in H₂-induced Anacystis. The enzyme apparently initiates a light-driven electron flow from H₂ to various low-potential acceptors including endogenous ferredoxin.     

Light and dark reactions of the uptake hydrogenase in anabaena 7120

Reactions of the uptake hydrogenase from Anabaena 7120 (A.T.C.C. 27893, Nostoc muscorum) were examined in whole filaments, isolated heterocysts, and membrane particles. Whole filaments or isolated heterocysts that contained nitrogenase consumed H₂ in the presence of C₂H₂ or N₂ in a light-dependent reaction. If nitrogenase was inactivated by O₂ shock, filaments catalyzed H₂ uptake to an unidentified endogenous acceptor in the light. Addition of NO₃⁻ or NO₂⁻ enhanced these rates. Isolated heterocysts consumed H₂ in the dark in the presence of electron acceptors with positive midpoint potentials, and these reactions were not enhanced by light. With acceptors of negative midpoint potential, significant light enhancement of H₂ uptake occurred. Maximum rates of light-dependent uptake were approximately 25% of the maximum dark rates observed. Membrane particles prepared from isolated heterocysts showed similar specificity for electron acceptors. These particles catalyzed a cyanide-sensitive oxyhydrogen reaction that was inactivated by O₂ at O₂ concentrations above 2%. Light-dependent H₂ uptake to low potential acceptors by these particles was inhibited by dibromothymoquinone but was insensitive to cyanide. In the presence of O₂, light-dependent H₂ uptake occurred simultaneously with the oxyhydrogen reaction. The pH optima for both types of H₂ uptake were near 7.0. These results further clarify the role of uptake hydrogenase in donating electrons to both the photosynthetic and respiratory electron transport chains of Anabaena.     

Direct Spectrophotometric Measurement of Photosystem I and Photosystem II Activities of Photosynthetic Membrane Preparations from Cyanophora paradoxa, Phormidium laminosum, and Spinach

Vesicles prepared with the French press from membranes of cyanelles of Cyanophora paradoxa retain O₂ evolution activity with rates up to 500 micromoles 2,6-dichlorophenolindophenol reduced per hour per milligram chlorophyll. This activity is immediately lost when the vesicles are transferred from the sucrose-phosphate-citrate preparation buffer into dilute phosphate buffer. Similar preparations from Phormidium laminosum, a thermophilic cyanobacterium retain activity under such conditions. Photosystem I activities of both cyanobacterial vesicle preparations were determined by direct spectrophotometric measurement of N,N,N′,N′-tetramethyl-p-phenylenediamine photooxidation in the presence of anthraquinone-2-sulfonate. The rates so determined were compared with rates of O₂ taken up in the presence of methyl viologen or anthraquinone-2-sulfonate as electron acceptors. The predicted stoichiometry of two was observed for moles of N,N,N′,N′-tetramethyl-p-phenylenediamine oxidized per mole of oxygen taken up. Anthraquinone-2-sulfonate was the better electron acceptor, and maximal rates of 943 micromoles per hour per milligram chlorophyll for O₂ uptake were observed for Phormidium laminosum preparations in the presence of superoxide dismutase. For purposes of comparison, spinach chloroplasts were assayed for similar activities. All preparations were readily assayed for photosystem I activity by the direct spectrophotometric method, which has advantages of simplicity and freedom from errors introduced by photoxidation of other substrates by photosystem I when O₂ uptake is measured.     

Engineering Pyranose 2-Oxidase for Modified Oxygen Reactivity

Pyranose 2-oxidase (POx), a member of the GMC family of flavoproteins, catalyzes the regioselective oxidation of aldopyranoses at position C2 to the corresponding 2-ketoaldoses. During the first half-reaction, FAD is reduced to FADH₂ and reoxidized in the second half-reaction by reducing molecular oxygen to H₂O₂. Alternative electron acceptors including quinones, radicals or chelated metal ions show significant and in some cases even higher activity. While oxygen as cheap and abundantly available electron acceptor is favored for many processes, reduced oxygen reactivity is desirable for some applications such as in biosensors/biofuel cells because of reduced oxidative damages to the biocatalyst from concomitant H₂O₂ production as well as reduced electron “leakage” to oxygen. The reactivity of flavoproteins with oxygen is of considerable scientific interest, and the determinants of oxygen activation and reactivity are the subject of numerous studies. We applied site-saturation mutagenesis on a set of eleven amino acids around the active site based on the crystal structure of the enzyme. Using microtiter plate screening assays with peroxidase/2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) and 2,6-dichlorophenolindophenol, variants of POx with decreased oxidase activity and maintained dehydrogenase activity were identified. Variants T166R, Q448H, L545C, L547R and N593C were characterized with respect to their apparent steady-state constants with oxygen and the alternative electron acceptors DCPIP, 1,4-benzoquinone and ferricenium ion, and the effect of the mutations was rationalized based on structural properties.     

Production of reactive oxygen species in decoupled, Ca2+-depleted PSII and their use in assigning a function to chloride on both sides of PSII

Extraction of Ca²⁺ from the oxygen-evolving complex of photosystem II (PSII) in the absence of a chelator inhibits O₂ evolution without significant inhibition of the light-dependent reduction of the exogenous electron acceptor, 2,6-dichlorophenolindophenol (DCPIP) on the reducing side of PSII. The phenomenon is known as “the decoupling effect” (Semin et al. Photosynth Res 98:235–249, 2008). Extraction of Cl^? from Ca²⁺-depleted membranes (PSII[–Ca]) suppresses the reduction of DCPIP. In the current study we investigated the nature of the oxidized substrate and the nature of the product(s) of the substrate oxidation. After elimination of all other possible donors, water was identified as the substrate. Generation of reactive oxygen species HO, H₂O₂, and O ₂ ^·? , as possible products of water oxidation in PSII(–Ca) membranes was examined. During the investigation of O ₂ ^·? production in PSII(–Ca) samples, we found that (i) O ₂ ^·? is formed on the acceptor side of PSII due to the reduction of O₂; (ii) depletion of Cl^? does not inhibit water oxidation, but (iii) Cl^? depletion does decrease the efficiency of the reduction of exogenous electron acceptors. In the absence of Cl^? under aerobic conditions, electron transport is diverted from reducing exogenous acceptors to reducing O₂, thereby increasing the rate of O ₂ ^·? generation. From these observations we conclude that the product of water oxidation is H₂O₂ and that Cl^? anions are not involved in the oxidation of water to H₂O₂ in decoupled PSII(–Ca) membranes. These results also indicate that Cl^? anions are not directly involved in water oxidation by the Mn cluster in the native PSII membranes, but possibly provide access for H₂O molecules to the Mn₄CaO₅ cluster and/or facilitate the release of H⁺ ions into the lumenal space.     

Characteristic temperature dependences of respiratory and photosynthetic electron-transport activities in membrane preparations from Anacystis nidulans grown at different temperatures

Electron-transport activities supported by seven different electron donor/acceptor couples in the light and in the dark, respectively, were measured in particle preparations of the cyanobacterium (blue-green alga) Anacystis nidulans after growth at 40, 30 and 25°C. The Arrhenius plots of the photosynthetic electron-transport reactions between ascorbate (plus 2,6-dichlorophenolindophenol (DCIP)) and NADP⁺, diphenylcarbazide and DCIP, diaminodurene and benzyl viologen (O₂), and the plot of the photooxidation of reduced horse heart cytochrome c showed a single discontinuity at approx. 24–25, 15–17 and 10–13°C in membranes derived from cells grown at 40, 30 and 25°C, respectively. By contrast, the dark respiratory electron-transport reactions between NADPH, ascorbate (plus DCIP) or reduced horse heart cytochrome c and oxygen, and the reduction by horse heart cytochrome c of the aa₃-type terminal oxidase as followed directly by dual-wavelength spectrophotometry, all gave Arrhenius plots distinguished by two distinct breaks: The break at the higher temperature corresponded to the break also found in the Arrhenius plots of the photosynthetic reactions while an additional discontinuity was observed at 17–18, 8–9 and 5–6°C in membranes prepared from cells grown at 40, 30 and 25°C, respectively. The temperatures at which the discontinuities in the Arrhenius plots occurred depended on the temperature at which the cells had been grown; they were independent, however, of the specific electron donors and acceptors employed. The characteristic features in the Arrhenius plots of respiratory and photosynthetic electron-transport reactions are discussed in terms of lipid-phase transitions in the cytoplasmic and the intracytoplasmic (thylakoid) membranes of A. nidulans. Implications for possibly distinct sites of the respiratory and photosynthetic electron-transport systems in A. nidulans will be mentioned.     

Generation of hydrogen peroxide on oxidation of NADH by hepatic plasma membranes

The oxidation of NADH by mouse liver plasma membranes was shown to be accompanied by the formation of H₂O₂. The rate of H₂O₂ formation was less than one-tenth the rate of oxygen uptake and much slower than the rate of reduction of artificial electron acceptors. The optimum pH for this reaction was 7.0 and theK _m value for NADH was found to be 3×10^–6 M. The H₂O₂-generating system of plasma membranes was inhibited by quinacrine and azide, thus distinguishing it from similar activities in endoplasmic reticulum and mitochondria. Both NADH and NADPH served as substrates for plasma membrane H₂O₂ generation. Superoxide dismutase and adriamycin inhibited the reaction. Vanadate, known to stimulate the oxidation of NADH by plasma membranes, did not increase the formation of H₂O₂. In view of the growing evidence that H₂O₂ can be involved in metabolic control, the formation of H₂O₂ by a plasma membrane NAD(P)H oxidase system may be pertinent to control sites at the plasma membrane.     

Engineering of dye-mediated dehydrogenase property of fructosyl amino acid oxidases by site-directed mutagenesis studies of its putative proton relay system

The flavoenzyme fructosyl amino acid oxidase (FAOD) catalyzes the oxidative deglycation of fructosyl amino acids, model compounds of glycated proteins. The high oxygen reactivity of FAODs limits their potential utility in amperometric enzyme sensors employing artificial electron mediators. To alter their electron acceptor availability, site-directed mutagenesis was carried out on conserved residues predicted to be involved in the proton relay system (PRS) of two eukaryotic FAODs, the FAOD from the marine yeast Pichia sp. N1-1 and amadoriase II from the fungus Aspergillus fumigatus. The substitution of a single conserved Asn residue in the putative PRS, Asn47Ala of N1-1 FAOD and Asn52Ala of amadoriase II, resulted in significant loss in the catalytic ability to employ O₂ as the electron acceptor, while having little effect on the dye-mediated dehydrogenase activity employing artificial electron acceptors instead of O₂.     

Depletion of Photosystem II-extrinsic proteins. I. Effects on O2- and N2-flash yields and steady-state O2 evolution

Wheat O₂-evolving Photosystem II (PS II) membranes having a PS II unit of approx. 200 chlorophylls (Chl), approx. 4 Mn/200 Chl, less than 1 P-700/3000 Chl and an electron-acceptor pool of approx. 2.5 equiv./PS II were analyzed and compared with wheat PS II membranes depleted (at least 90%) of the 17 and 23 kDa proteins by NaCl extraction during Triton X-100 isolation of membranes. Extraction of these proteins caused approx. 50% decrease in O₂ evolution in any light regime and an increase of approx. 2 equiv./PS II of the electron-acceptor pool, but affected neither Mn abundance, photoreduction of DCIP by tetraphenylboron, or N₂ yield (from NH₂OH) from a single flash. Mass spectrometric analyses of O₂ flash yields in the presence of potassium ferricyanide showed that both chloroplasts and the unextracted PS II membranes yielded oscillations compatible with S₀/S₁/S₂/S₃ of 25:75:0:0 and α (0.1) and β (0.05). Depletion of 17 and 23 kDa proteins resulted in a two-fold increase in α, approx. 25–40% disconnection of the S state complex from the PS II trap complex but with no change in β. Preincubation of control or extracted PS II membranes with potassium ferricyanide permitted a significant double-hit on the first flash. In the absence of an added electron acceptor, N₂ flash yields were more sustained with 17 and 23 kDa depleted than with 17 and 23 kDa sufficient PS II membranes. In contrast, no significant O₂ flash yields were observed with extracted PS II preparations under these conditions (control PS II membranes showed a predictable O₂ pattern before damping after only 5–6 flashes). These results suggest that extraction of the 17 and 23 kDa proteins results in an increase of pool size on the PS II acceptor side (seen as unmasking ‘Component C’). ‘Component C’ can mediate electron transfer from Q⁻ to Z⁺ (S₂).     

Inhibitory effect of molecular hydrogen on C2H2-reducing activity in Anabaena 7120 preilluminated in red or blue light

Molecular hydrogen inhibits nitrogenase activity in Anabaena pre-illuminated with red or blue light. The inhibitory effect of molecular hydrogen decreased in the presence of oxygen and several electron acceptors. When NH₄Cl and urea were added simultaneously with molecular hydrogen, marked synergistic inhibitory effects took place. The inhibitory effect of molecular hydrogen disappeared or was weakened after the suppression of hydrogenase activity. The addition of O₂ and electron acceptors to systems showed no enhancing effect on the C₂H₂-reducing activity.     

Characterization of pyranose oxidase variants for bioelectrocatalytic applications

Pyranose oxidase (POx) catalyzes the oxidation of d-glucose to 2-ketoglucose with concurrent reduction of oxygen to H₂O₂. POx from Trametes ochracea (ToPOx) is known to react with alternative electron acceptors including 1,4-benzoquinone (1,4-BQ), 2,6-dichlorophenol indophenol (DCPIP), and the ferrocenium ion. In this study, enzyme variants with improved electron acceptor turnover and reduced oxygen turnover were characterized as potential anode biocatalysts. Pre-steady-state kinetics of the oxidative half-reaction of ToPOx variants T166R, Q448H, L545C, and L547R with these alternative electron acceptors were evaluated using stopped-flow spectrophotometry. Higher kinetic constants were observed as compared to the wild-type ToPOx for some of the variants. Subsequently, the variants were immobilized on glassy carbon electrodes. Cyclic voltammetry measurements were performed to measure the electrochemical responses of these variants with glucose as substrate in the presence of 1,4-BQ, DCPIP, or ferrocene methanol as redox mediators. High catalytic efficiencies (I_max^app/K_M^app) compared to the wild-type POx proved the potential of these variants for future bioelectrocatalytic applications, in biosensors or biofuel cells. Among the variants, L545C showed the most desirable properties as determined kinetically and electrochemically.     

Novel Physiological Features of Carboxydothermus hydrogenoformans and Thermoterrabacterium ferrireducens

Carboxydothermus hydrogenoformans is able to grow by conversion of CO to H₂ and CO₂. Besides CO, only pyruvate was described as serving as an energy source. Based on 16S rRNA gene sequence similarity, C. hydrogenoformans is closely related to Thermoterrabacterium ferrireducens. T. ferrireducens is like C. hydrogenoformans a gram-positive, thermophilic, strict anaerobic bacterium. However, it is capable of using various electron donors and acceptors for growth. Growth of C. hydrogenoformans with multiple electron donors and acceptors was tested. C. hydrogenoformans oxidized formate, lactate, glycerol, CO, and H₂ with 9,10-anthraquinone-2,6-disulfonate as an electron acceptor. Sulfite, thiosulfate, sulfur, nitrate, and fumarate were reduced with lactate as an electron donor. T. ferrireducens oxidized CO with 9,10-anthraquinone-2,6-disulfonate as an electron acceptor but did not produce H₂ from CO. In contrast to what was published before, T. ferrireducens was able to grow on lactate with sulfite, sulfur, and nitrate as electron acceptors.